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Learnings and Recommendations from Four EU-ToxRisk Case Studies on Applying New Approach Methodologies Data to Support Read-Across
Susanne Hougaard BennekouThe National Food Institute The Danish Technical [email protected]
Conflict of Interest Statement
No conflicts of interest
Overview/Objectives
To provide examples on how New Approach Methodologies (NAM) can be used in read-across
To provide learnings and recommendations on – Reporting– Performance of NAMs– Weight of Evidence
EU-ToxRisk
An Integrated EUropean “Flagship” Program Driving Mechanism-based Toxicity Testing and Risk Assessment for the 21st Century1. Improved toxicological knowledge and quantitative understanding of concentration- and time-response relationships for key RDT and DART adverse outcomes to be annotated in semi-qAOP and some qAOP. 2. New robust Read-across procedures incorporating toxicokinetics data and similarity evaluation on the level of KE activation, and a complementary KE screen battery to easily fill data gaps of the Read-across procedure and meet regulatory needs. 3. A validated set of tiered IATAs for ab initio RDT and DART assessment for regulatory use. 4. Novel validated in silico tools and in vitro test systems ready….and more, see http://www.eu-toxrisk.eu/
RDT
selection of test systemscase studies
biokineticstranscriptomics cytotoxicity
chemical selection
quantitative key event
IATAStrategies
Expl
oita
tion
Risk
ass
essm
ent
in silico and experimental ADME
computational modelling
concentrations
Liver Kidney Lung Neuro DART
HepG2 + CYPs HepaRG
semi3Dsandw.
PHH
multicell3D PHH
3D hRPTEC
iPSCsensoryneurons
humanEST-
cardiacneuro
zebrafish
embryo
3D air/liquid
hLEC
2DhRPTEC
primaryhRPTECT
iPSC 3D
floatingneurons
neuritegrowthassay
2D hLEC
primaryA/L
3D hLEC
2DSH-SY5Y LUHMES
DART
22D and 3D high throughput and high content pathway toxicity reporters
in silico prediciton of ADME and toxicity; biokinetics data
2translation to fresh human tissue, patient samples and human genetics
tiere
d t
estin
g
Matrix
Read Across rarely accepted by regulatory authorities
• Based often only on structural & physicochemical data no biological proof for similarity
• Lack of sufficient evidence to substantiate read-across justifications- fail to demonstrate toxicokinetic
• and toxicodynamic similarities Including lack of data on analogues provided in dossier
•• Lack of scientific plausibility
Disagreement with hypothesis, data not supportive of arguments presented, high uncertainty coupled with lack of evidence
Read-Across: Current Limitations
Basis for Learnings: Case Studies
Prediction of a 90 day Repeated Dose Toxicity Study for 2-Ethylbuturic acid using a read-across approach to other
branched carboxylic acids
Read-across based filling of DART data-gap for methyl hexanoic acid using NAM
Mitochondrial complex-III-mediated neurotoxicity of
azoxystrobin? Read-across to other strobilurins
Identification and characterisation of parkinsonian hazard liability of
deguelin by an AOP-based testing and read across approach
Case Studies – Examples of NAMs
Prediction of a 90 day Repeated Dose Toxicity Study for 2-Ethylbuturic acid using a read-across approach to other
branched carboxylic acids
Case Studies – Examples of NAMs
Read-across based filling of DART data-gap for methyl hexanoic (MHA) acid using
NAMZebrafish Embryo Test (ZET)
ZET CHA reporter (cranio-facial differentiation) (ZET Reporter)
Human neural stem cell differentiation model (UKN1)
Mouse embryonic stem cell differentiation model (mEST)
CALUX reporter assay panel (CALUX)
In vivo Oral Equivalent Doses mouse
Data integration statistical models
Dempster-Schaefer Theory approach
Bayesian Automatic Classification approach
Biological Fingerprint Classification approach
Case Studies – Examples of NAMsCI (5XTD) DeguelinRotenoneIdentification and characterisation
of parkinsonian hazard liability of deguelin by an AOP-based testing
and read across approach
Case Studies – Examples of NAMs
Mitochondrial complex-III-mediated neurotoxicity of
azoxystrobin? Read-across to other strobilurins
Mathias Hertzler, BfR – ChairAndrea Terron, EFSA Joop de Knecht, RIVMBodo Hass, BfArM Richard Judson, US-EPADerek Knight, ECHA J.W. van der Laan, CBG MEBIvan Drobev, BAUA Leon van Aerts, CBG MEBDominique Masset, ANSM Lidka Maslankiewicz, RIVMElke Roehrdanz, BfArM Magdalini Sachana, OECDEmiel Rorije, RIVM, Martin Paparella, IMUGeorge Kass, EFSA Sankalp Jain, UNIVIEJanine Ezendam, RIVMSuzanne Fitzpatrick, FDATewes Tralau, BfRThomas Steger-Hartmann, BayerUlrike Bernauer, BfRWeida Tong, FDASankalp Jain, UNIVIEbranched carboxylic acids
The EU Regulatory Advisory Board
1. Get the science right2. Convince the regulators that you got the
science right3. Address the specific requirements of the
regulatory process
Aimed at Regulators
Aimed at Scientist
1. Fulfills all requirements of GD2112. Guides the user concerning the types of
answers and detail of information required3. Includes acceptance criteria for test
elements4. Defines the cells sufficiently and
transparently
A database containing exemplary descriptions of more than 20 cell-based tests.
Regulatory Feedback
1. AOP-based testing strategy• Test all KEs?• Test KEs with different assays?
2. If no AOP available/AOP weak: Describe the scientific rational of the testing in more detail
3. Necessary to address other AOPs/MoAs relevant for the problem formulation
Learnings - AOPs
Overall Learnings
1. Helpful to include description of results with reference compounds even though they are not part of the Read-Across
2. PBPK modelling is useful – although there is still some skepticism3. Useful to include compounds in a category approach which do not have in vivo data4. An analogue approach based on one compound can be justified5. Difficult to conclude that a category is adequate: structural similarity, number of source
chemicals and their responses. In practice the number of source chemicals with in vivo data will be low
6. Case studies have shown some validity to biological read-across7. Even with a reporting template tailored for the purpose – no consensus on the level of
reporting8. Situations where current in vivo models have limitations – easier to waive a study based on a
clear mechanistic hypothesis9. Validity of non-guideline test needs detailed description
Further Guidance would be helpful on • On reporting of receptor docking/modelling• How much evidence is needed to show low toxicity? -> A clear trigger can define space around a
negative prediction,• Data on reference compounds• Does all KEs in an AOP need to be tested? Is It OK with just a few if they show a consistent
pattern?• Test method description, performance and validation – refer possibly to EU-ToxRisk Toxtemp
(Krebs et al 2019) ?• On how to integrate many lines of evidence? What integrating methodology to apply and when;
When is a prediction from such methodology adequate?• What does biologically RAX need to address?
Overall Learnings
Read-Across Assessment Steps – the EUToxRisk Approach:Making use of New Approach Methodologies (NAMs)
Read-Across Assessment Steps – the EU-ToxRisk Approach
Experimental NAM data
In silico toolsfor integrationof NAM data
SummaryFor a successful regulatory read-across:• (Currently) no read-across without testing
– Regulatory authorities demand standard OECD guideline testing• For application of non-guideline in vitro methods
– Ensure transparency and quality documentation– Explain methodology– Justify the read-across hypothesis with good data and scientific
evidence– Integrate hazard & kinetic data and assess limitations
Advisory Document on read-across based on data from new approach methodologies (NAMs)” Publication of case studies on OECD web-site
References• Escher SE et al. Towards grouping concepts based on new approach methodologies
in chemical hazard assessment: the read-across approach of the EU-ToxRisk project. Arch Toxicol. 2019 Dec;93(12):3643-3667. doi: 10.1007/s00204-019-02591-7
• Krebs A et al. Template for the description of cell-based toxicological test methods to allow evaluation and regulatory use of the data. ALTEX. 2019;36(4):682-699. doi: 10.14573/altex.1909271.
• Terron A et al. An adverse outcome pathway for parkinsonian motor deficits associated with mitochondrial complex I inhibition. Arch Toxicol. 2018 Jan;92(1):41-82. doi: 10.1007/s00204-017-2133-4.
• Ball N et al. Toward Good Read-Across Practice (GRAP) guidance. ALTEX. 2016;33(2):149-66. doi: 10.14573/altex.1601251.
• Rovida C et al. Internationalization of read-across as a validated new approach method (NAM) for regulatory toxicology. ALTEX. 2020 Apr 30. doi: 10.14573/altex.1912181.
Acknowledgements
Partners of the EU-ToxRisk Consortium Regulatory Advisory Board of EU-ToxRisk
Case study leaders: Sylvia Escher, ITEM-Frauenhofer, Germany Bob van der Water, Leiden University, Netherlands Dinant Kroese, TNO, Netherlands Hennicke Kamp, BASF, Germany
Funding:European Union’s Horizon 2020 research and innovation programme EU-ToxRisk (grant agreement No 681002)